Glucocorticoids are a class of hormones produced by the adrenal cortex and involved in metabolism and immunity. Since glucocorticoids have strong anti-inflammatory effects and immunosuppressive effects, artificially synthesized glucocorticoids have been used as drugs for treating allergic diseases and autoimmune diseases.
Furthermore, since glucocorticoids have growth inhibitory effects on lymphocytes that have become cancerous, artificially synthesized glucocorticoids have been used in chemotherapy for lymphoma or lymphocytic leukemia. Since it is quite rare for the administration of glucocorticoids to produce side effects such as myelosuppression or severe gastrointestinal symptoms, glucocorticoids are essential drugs for treating lymphoma or lymphocytic leukemia.
Glucocorticoids have been used for the treatment of various diseases. However, there are cases in which some patients exhibit resistance to glucocorticoids, and glucocorticoids do not exert effects in such cases. It is reported that from 5% to 10% of the patients with asthma, about 30% of the patients with rheumatic diseases, from 20% to 50% of the patients with inflammatory bowel diseases, and from 10% to 25% of the patients with childhood acute lymphocytic leukemia have resistance to glucocorticoids. Therefore, development of a technique for canceling resistance to glucocorticoids has been requested.
The following findings have been reported with regard to glucocorticoid receptors (GRs).
GRs, when bound to glucocorticoids, function as transcription factors in nuclei. The base sequence of a human GR gene is known (NR3C1, Gene ID: 2908, NCBI Reference Sequence: NC—000005.9). Nine exons are present in the human GR gene, and two splicing variants, GRα and GRβ, are generated by alternative splicing within exon 9 (having 4,111 bases).
GRα is a protein having 777 amino acid residues translated from a mature mRNA formed by joining together of exons 1 to 9. GRα translocates into the nucleus in a ligand-dependent manner and functions as a transcription factor.
GRβ is a protein having 742 amino acid residues translated from a mature mRNA formed by joining together of exons 1 to 8 and a part of exon 9 (from 2631st base to 4111th base from the 5′-end of exon 9). GRβ lacks a part of the ligand-binding domain, and has no ligand-binding capacity. In the nucleus, GRβ competitively antagonizes GRα to which a ligand is bound, and inhibits the transcription factor activity of GRα.
With regard to glucocorticoid resistance of cells, the following reports have been made.
It has been reported that, in cell lines (CL-1 cells, GL-1 cells) derived from canine lymphoma or leukemia, glucocorticoid resistance is canceled by inhibiting the function of nuclear factor-κB (NF-κB), and cell proliferation is inhibited by the addition of a glucocorticoid (for example, see Document 1). This suggests that the inhibition of NF-κB function increases GR expression levels in the cells, thereby making it easier for the cell proliferation inhibitory effects of glucocorticoids to be exerted on the cells.
It has also been reported that, in Raji cells derived from human Burkitt's lymphoma and peripheral blood-derived cells from patients with acute lymphocytic leukemia, glucocorticoid resistance is canceled, and GRα expression levels are increased, by inhibiting NF-κB function using an siRNA (for example, see Document 2). This result suggests the possibility that glucocorticoid resistance is canceled by increased expression of GRα in the cells.
With regard to GR expression, the following reports have been made.
It has been reported that, in a colon cancer cell line (HT-29 cells) and a breast cancer cell line (MCF-7 cells), histone deacetylase inhibitors such as trichostatin A and sodium butyrate and a DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine increase GRα expression levels while decreasing GRβ expression levels (for example, see Document 3). At the same time, the expression level of ASF/SF2, which is a type of serine/arginine rich protein (SR protein), increases, suggesting the possibility that ASF/SF2 may be involved in the regulation of the splicing of the GR mRNAs. SR proteins are splicing factors, and regulate the splicing of pre-mRNAs into mature mRNAs in the nucleus.
It has been reported that a study comparing mRNA levels in peripheral lymphocytes from 28 healthy volunteers reveals the negative correlation between the expression level of serine/arginine-rich protein 30c (SRp30c), which is a type of SR protein, and the ratio of the GRα expression level to the GRβ expression level (GRα/GRβ) (for example, see Document 4).
It has also been reported that, in a neutrophil-like cell line (retinoic acid-stimulated PLB-985 cells), knocking-down of SRp30c using antisense oligonucleotides results in a decrease in the expression levels of the GRβ mRNA and an increase in the expression levels of the GRα mRNA (for example, see Document 5).
In addition, it was found that a 5-base sequence of AGGAC is present in the GR gene at relatively high frequency, and this sequence is expected to be an SRp30c recognition sequence (for example, see Document 6).
These findings suggest that the alternative splicing within exon 9 of the GR gene is regulated by SRp30c, which is a type of SR protein. That is, the above findings suggest that a site to which SRp30c binds is present in exon 9 of the pre-mRNA of GR, and that SRp30c binds to this site and regulates the splicing of the pre-mRNA of GR into the mature mRNA of GRβ.
However, there are no reports about whether or not the glucocorticoid sensitivity of cells is actually altered by inhibiting SRp30c function, and this point is unclear.
Meanwhile, a technique for inhibiting a function of an SR protein by inhibiting the phosphorylation of the SR protein is disclosed. For example, a technique is disclosed (for example, see Documents 7 and 8) in which an isonicotinamide compound SRPIN 340, which is an inhibitor of an SR protein kinase, is used as an antiviral agent.
Document 1: Matsuda A, et al. Res. Vet. Sci., 2010, 89(3):378-382.
Document 2: Matsuda A, et al. The 14th International Congress of Immunology, August 2010, Volume 22, Supplement number 1, p. v8.
Document 3: Piotrowska H, et al. Arch. Med. Res., 2009, 40: 156-162.
Document 4: Watanuki T, et al. J. Affect. Disord., 2008, 110(1-2): 62-69.
Document 5: Xu Q, et al. J. Biol. Chem., 2003, 278: 27112-27118.
Document 6: Paradis C, et al. RNA, 2007, 13: 1287-1300.
Document 7: Karakama Y, et al. Antimicrob. Agents Chemother., 2010, 54(8): 3179-318.
Document 8: International Publication No. WO 2005/063293.